PROJECT SUMMARY In nature, all cells and most biological macromolecules are decorated with sugars. These sugar modifications, which are referred to as ?glycans?, modulate and mediate a wide array of cellular processes. Not surprisingly, aberrant glycan structures have been implicated in a variety of human diseases. The study of glycan structure and function ? known as the field of glycomics ? is a burgeoning field, which promises to greatly enhance our understanding of human health and disease. A significant challenge facing glycomics is to understand the function of the enormous repertoire of glycan determinants within the human glycome, which has been estimated to be many thousands. This challenge is compounded by the deficit of readily accessible glycan recognition molecules (GRMs) for these determinants. Just as high affinity reagents for proteins have been a boon to the development of proteomics, access to GRMs are anticipated to enable researchers to test hypotheses regarding specific glycan structure and function. The repository of GRMs for the human glycome is extremely limited. Furthermore, many GRMs, especially lectins, have been shown to bind to multiple glycan structures, and commercial preparations of lectins have demonstrated great inconsistencies in binding affinities. These issues have stimulated research in developing alternative selective and high-affinity GRMs that exceed the performance of traditional GRMs. Despite their promise, many of these alternative technologies, including antibodies and nucleic acid aptamers, suffer from several shortcomings. In this proposal we aim to address the critical need for robust GRMs by developing a new class of GRMs based on the heteromultivalent display of chemical functionality along a stable ssDNA scaffold. Using our recently devised method for T4 DNA ligase-mediated sequence-specific incorporation of functional groups along ssDNA scaffolds, we will develop an in vitro evolution system to identify GRMs from large libraries of functionalized ssDNA (>10 members). In aim 1 we will qualify a high-fidelity codon to be used during in vitro evolution of GRMs. In aim 2, we will apply this platform to the discovery of functionalized ssDNA GRMs that recognize different biantennary glycan structures. We will then demonstrate that this approach enables the control of glycan specificity by iterated rounds of positive and negative in vitro evolution against different glycan targets.